Nanoscale Imaging of Proteins and Nucleic Acids via Expansion Microscopy

ABSTRACT

The invention enables in situ genomic and transcriptomic assessment of nucleic acids to be conducted in biological specimens that have been physically expanded. The invention leverages the techniques for expansion microscopy (ExM) to provide new methods for in situ genomic and transcriptomic assessment of nucleic in a new process referred to herein as “expansion fluorescent in situ hybridization” (ExFISH).

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Ser. No. 62/202,421, filed Aug. 7, 2015, the contents of which are incorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under 5-DPI-NS087724 awarded by NIH, Hertz Foundation, ODGE Lemelson & Viterbi, 5-DPI-N S087724 awarded by NIH and NSF. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Nanoscale-resolution imaging of RNA throughout cells, tissues, and organs is key for an understanding of local RNA processing, mapping structural roles of RNA, and defining cell types and states. However, it has remained difficult to image RNA in intact tissues with the nanoscale precision required to pinpoint associations with cellular compartments or proteins important for RNA function.

Expansion microscopy (ExM) enables imaging of thick preserved specimens with ˜70 nm lateral resolution. Using ExM the optical diffraction limit is circumvented by physically expanding a biological specimen before imaging, thus bringing sub-diffraction limited structures into the size range viewable by a conventional diffraction-limited microscope. ExM can image biological specimens at the voxel rates of a diffraction limited microscope, but with the voxel sizes of a super-resolution microscope. Expanded samples are transparent, and index-matched to water, as the expanded material is >99% water. The original ExM protocol worked by labeling biomolecules of interest with a gel-anchorable fluorophore. Then, a swellable polyelectrolyte gel was synthesized in the sample, so that it incorporated the labels. Finally, the sample was treated with a nonspecific protease to homogenize its mechanical properties, followed by dialysis in water to mediate uniform physical expansion of the polymer-specimen composite. All of the chemicals required for ExM can be purchased except for the gel-anchorable label, which requires custom synthesis and raises the barrier for researchers to adopt the method. Another drawback of the ExM protocol is that genetically encoded fluorophores cannot be imaged without antibody labeling. Additionally, ExM was unable to retain native proteins in the gel and used custom made reagents not widely available. Thus, it would be desirable to leverage ExM to devise new methods for in situ retention and imaging of nucleic acids and proteins within a sample.

SUMMARY OF THE INVENTION

A small molecule linker is synthesized that enables RNA to be covalently attached to the ExM gel. This method, referred to as ExFISH, enables RNA fluorescent in situ hybridization (FISH), which enables identification of transcripts in situ with single molecule precision. In RNA FISH, a set of fluorescent probes complementary to a target strand of mRNA are delivered^(2,3). Single molecule FISH (smFISH) can be performed with multiple fluorophores delivered to a single mRNA via oligonucleotide probes⁴. In intact tissues, amplification strategies, such as hybridization chain reaction (HCR)^(5,6), and branched DNA amplification^(7,8), can enable a large number of fluorophores to be targeted to a single mRNA. ExFISH can support smFISH in cell culture, and HCR-amplified FISH in intact mouse brain tissues. ExFISH can reveal nanoscale structures of long non-coding RNAs (lncRNAs), as well as for localizing neural mRNAs to individual dendritic spines. ExFISH will be useful for a diversity of questions relating the structure and location of RNA to biological functions.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided to the Office upon request and payment of the necessary fee.

FIG. 1A-1I: Design and validation of ExFISH chemistry. (1A) Acryloyl-X SE (top left) is reacted to LABEL-IT® amine (top right) via NETS-ester chemistry to form LabelX (middle), which serves to make RNA gel-anchorable by alkylating its bases (e.g., the N7 position of guanines) (bottom). (1B) Workflow for ExFISH: biological specimens are treated with LabelX (left), which enables RNA to be anchored to the ExM gel (middle). Anchored RNA can be probed via hybridization (right), after gelation, digestion, and expansion. (1C) smFISH image of ACTB before expansion. Inset shows zoomed-in region, highlighting transcription sites in nucleus. (1D) As in (1C), using ExFISH. (1E) smFISH counts before versus after expansion for seven different transcripts (n=59 cells; each symbol represents one cell). (1F) smFISH image of XIST long non-coding RNA (1ncRNA) in the nucleus of a HEK293 cell before expansion (white line denotes nuclear envelope in 1F-1H). (1G) As in (1F), using ExFISH. (1H) smFISH image before expansion (top), and using ExFISH (bottom), of NEAT1 lncRNA in the nucleus of a HeLa cell. Magenta and green indicate probesets binding to different parts of the 5′ (1-3756 nts) of NEAT1 (see Methods). (1I) Insets showing a NEAT1 cluster (boxed region of (1H)) with smFISH (left) and ExFISH (right). Scale bars (white, in pre-expansion units; blue scale bars are divided by the expansion factor noted))): (1C, 1D) 10 μm (expansion factor, 3.3×), inset 2 μm; (1F, 1G) 2 μm (3.3×), Z scale represented by color coding in pre-expansion units; (1H) 2 μm (3.3×); (1I) 200 nm (3.3×).

FIG. 2A-2E: Serially hybridized and multiplexed ExFISH. (2A) Widefield fluorescence image of ExFISH targeting GAPDH. (2B) Boxed region of (2A), showing 5 repeated re-stainings following probe removal (see Methods); lower right panel, overlay of the 5 images (with each a different color, red, green, blue, magenta, yellow), showing co-localization. (2C) ExFISH RNA counts for each round, normalized to the round 1 count; plotted is mean±standard error; n=3 regions of (2A). (2D) Signal-to-noise ratio (SNR) of ExFISH across the five rounds of staining of (2A), computed as the mean puncta brightness divided by the standard deviation of the background. (2E) Composite image showing ExFISH with serially delivered probes against six RNA targets in a cultured HeLa cell (raw images in FIG. 9); colors are as follows: NEAT1, blue; EEF2, orange; GAPDH, yellow; ACTB, purple; UBC, green; USF2, light blue. Scale bars (expanded coordinates): (2A) 20 μm; (2B) 10 μm; (2E) 20 μm.

FIG. 3A-3K: Nanoscale imaging of RNA in mammalian brain. (3A) Widefield fluorescence image of Thyl-YFP mouse brain. (3B) Post-expansion widefield image of (3A). (3C) Widefield fluorescence showing HCR-ExFISH of YFP mRNA in the sample of (3B). (3D) As in (3C), but for Gadl mRNA. (3E) Composite of (3B-3D), highlighting distribution of Gad1 versus Thy1-YFP mRNAs. (3F) Confocal image of mouse hippocampal tissue from (e) showing single RNA puncta. Inset, one plane of the boxed region (red, YFP protein; cyan, YFP mRNA; magenta, Gad1 mRNA). (3G(i)) Confocal image and (3G(ii)) processed image of HCR-ExFISH using a missense D1g4 probe, in Thyl-YFP mouse tissue (green, YFP protein). The raw image (3G(i)) uses alternating probes in two colors (red, D1g4 missense even; blue, D1g4 missense odd). The processed image (3G(ii)) shows zero co-localized spots (magenta). (3H, 3I) As in (3G(i) and 3G(ii)), but for HCR-ExFISH targeting Actb in Thy1-YFP mouse brain (green, YFP protein; red, Actb even, and blue, Actb odd in (3H(i)); co-localized spots in magenta (3H(ii))). (3I) Confocal image of hippocampal tissue showing co-localized D1g4 puncta (magenta) overlaid on YFP (green). (3J(i), 3J(ii)) Two representative examples of dendrites with D1g4 mRNA localized to spines (arrows). (3K(i), 3K(ii)) As in (3J), but with HCR-ExFISH of Camk2a mRNA showing transcripts in dendritic spines and processes. Scale bars (white, in pre-expansion units; blue scale bars are divided by the expansion factor noted): (3A) 500 μm; (3B-3E) 500 μm (expansion factor 2.9×); (3F) 50 μm (2.9×), inset 10 μm; (3G-3I) 10 μm (3×); (3J, 3K) 2 μm (3×). (3E, 3I) maximum-intensity projection (MIP) 27 μm thick (pre-expanded units); (3G, 3H, 3J, 3K) MIPs ˜1.6 μm thick.

FIG. 4A-4B: (4A) Epi-fluorescence image of single molecule FISH (smFISH) against GAPDH on HeLa cells expanded without LabelX treatment. (4B) Epi-fluorescence image of smFISH performed against GAPDH on expanded HeLa cells treated with LabelX. Images are maximum intensity projections of 3-D stacks. Nuclei stained with DAPI (shown in blue). Scale bars: 20 μm (post-expanded units).

FIG. 5A-5E: To assess the effect of LabelX on fluorescent in situ hybridization, fixed HeLa cells were stained with smFISH probe-sets, followed by DNAse I treatment to remove the staining. The cells were then treated with LabelX and stained again with the same smFISH probe-sets. (5A) UBC staining before LabelX treatment and (5B) UBC staining after probe removal and LabelX treatment. (5C) EEF2 staining before LabelX treatment. (5D) EEF2 staining after probe removal and LabelX treatment. (5E) Comparison of smFISH spots counted for individual cells before LabelX, and after probe removal and application of LabelX. The number of RNA molecules detected in a given cell was quantified using an automated spot counting algorithm (n=7 cells for each bar). Plotted are mean±standard error; no significant difference in spot counts before vs after LabelX (p>0.5 for before vs. after for UBC, p>0.5 for before vs. after for EEF2; t-test, unpaired, two-tailed). Images in 5A-5D are maximum intensity projections of 3-D stacks; scale bars: 10 μm (pre-expanded units).

FIG. 6A-6G: Different RNA species spanning 3 orders of magnitude in abundance were detected via single molecule RNA fluorescent in situ hybridization (FISH) in HeLa cells before and after ExM with LabelX treatment (shown in FIG. 1E). (1A) Ratio of FISH spots detected after expansion to spots detected before expansion for single cells. Representative before vs. after ExFISH images shown: (1B,1C) TFRC; (1D,1E) GAPDH; (1F, 1G) ACTB. Scale bars, 10 μm (pre-expanded units) in 1B, 1D, 1F; 1C, 1E, 1G, expanded physical size 21 μm (imaged in PBS).

FIG. 7A-7E: (7A) Pre-expansion widefield image of a cultured HeLa cell stained with DAPI to visualize the nucleus (top panel) and smFISH probes against ACTB (bottom panel). (7B) Post-expansion widefield image of the same cell as in (a). (7C) Pre-expansion widefield image of LabelX treated Thyl-YFP brain slice (left panel, YFP protein) stained with DAPI (right panel) (MIP, 4 μm z-depth). (7D) Post-expansion image of the same region as in (c) (MIP, 12 μm). (7E) Ratio of the expansion factor of cell bodies for individual cells to the expansion factor of their respective nuclei. smFISH stain is used to outline the boundaries of the cell bodies of cultured cells while the endogenous YFP protein is used to demarcate the cell bodies of neurons in Thy1-YFP brain slices. Plotted are mean±standard error. The ratio for both cultured cells and brain slices did not significantly deviate from one (p>0.05 for both, 1-sample t-test; n=6, cultured HeLa cells; n=7, cells in 1 brain slice). Scale bars, 10 μm.

FIG. 8A-8D: (8A) Representative FISH image of TOP2A in a single HeLa cell before expansion (MIP of cell thickness). (8B) ExFISH image of cell in (8A) taken with the same optical parameters. (8C) Merged image of (8A) and (8B) (red and green for before and after expansion respectively); distance measurements between pairs of mRNA spots before (L, red line) and after (L′, green line; note that these lines overlap nearly completely) expansion were used to quantify expansion isotropy. (8D) Mean of the absolute value of the measurement error (i.e., |L-L′|) plotted against measurement length (L) for all pairs of mRNA spots (mean±standard deviation, N=4 samples, 6.8×10⁵ measurements). Scale bars: white, 10 μm pre-expansion units; blue, white scale bar divided by expansion factor. Orange line indicates diffraction limit of the microscope used (see methods for details).

FIG. 9A-9B: (9A) Five consecutive widefield fluorescence images (top to bottom, then left to right) of GAPDH, applied to the cell of FIG. 2a . (9B) Widefield fluorescence images showing ExFISH with serially delivered probes against six RNA targets (right to left, then top to bottom: NEAT1, EEF2, ACTB, UBC, GAPDH, and USF2) in a cultured HeLa cell (raw images of composite shown in FIG. 2E). Scale bars: 20 μm in expanded units.

FIG. 10: FISH probes bearing HCR initiators are hybridized to a target mRNA. During amplification, metastable DNA hairpins bearing fluorophores assemble into polymer chains onto the initiators, thus amplifying signal downstream of the FISH probe hybridization event.

FIG. 11A-11C: (11A) Widefield image of a LabelX treated Thy1-YFP brain slice (YFP protein, green) stained with probes against YFP (red) and Gad1 (magenta) followed by HCR amplification. Probes against YFP transcripts were amplified with the B1 amplifier set (see Methods) while probes against Gad1 transcripts were amplified with the B2 amplifier set (MIP, 59 μm). (11B) Widefield image of LabelX treated Thy1-YFP brain slice (YFP protein, green) treated with the same HCR amplifiers as in (a) (namely B1 (red) and B2 (magenta)) without the addition of probes (MIP, 50 μm). (11C) HCR spots detected per volume of expanded sample. Analysis was performed on samples which were either treated or not treated with FISH probes followed by HCR amplification. An automated spot counting algorithm was used to count HCR spots. The endogenous YFP protein was used to delineate regions used for the analysis. Plotted are mean±standard error. HCR spot counts are significantly different in the presence of probes than without probes (p<0.05 for both B1 and B2 amplifier sets, Welch's t-test; n=4 fields of view each). Scale bars: 50 μm.

FIG. 12A-12C: (12A) Volume rendering of Thy1-YFP (green) brain tissue acquired by lightsheet microscopy with HCR-ExFISH targeting YFP (red) and Gad1 (blue) mRNA. (12B) A maximum intensity projection (˜8 μm in Z) of a small subsection of the volume, showing the high resolution of imaging and single molecule localization of imaging expanded specimens with lightsheet imaging (scale bar: 10 μm, in pre-expansion units, expansion factor, 3×). (12C) Zoom in of the volume rendering in (12A) (scale bar: 20 μm, in pre-expansion units, 3×).

FIG. 13A-13G: (13A) Schematic showing two color amplification of the same target. A transcript of interest is targeted by probes against alternating parts of the sequence, and bearing two different HCR initiators, allowing for amplification in two colors. (13B) Confocal image showing FISH staining with HCR amplification against the Camk2a transcript in two colors (red and blue; YFP fluorescence shown in green). (13C) The result of an automated two-color spot co-localization analysis performed on the data set shown in (13B). Each purple spot represents a positive co-localization identified by the algorithm and overlaid on the confocal image of YFP. (13D, 13E) Zoom in of dendrites showing two color FISH staining with HCR amplification against Camk2a transcripts. (13F, 13G) As in (13D, 13E) but against D1g4 transcripts. Top row shows the raw two color staining data corresponding to the bottom row showing co-localized spots identified by the automated algorithm. Scale bars: (13B, 13C) 10 μm (3×); (13D-13G) 2 μm (3×). (13B-13G) are MIP of ˜1.6 μm thickness in unexpanded coordinates.

FIG. 14A-14B: (14A) Schematic for HCR amplification and reversal. HCR amplification is initiated with custom-made HCR hairpins bearing toe-holds for toe-hold mediated strand displacement. After amplification, the addition of a disassembling strand initiates the disassembly of the HCR polymers via strand displacement. (14B) ExFISH-treated Thy1-YFP brain slice (YFP in blue) is shown stained with YFP FISH probes bearing HCR initiators and amplified with custom made HCR hairpins bearing toe-holds for strand displacement (green dots). The different panels show the state of HCR reversal at different times after the addition of strands to initiate the disassembly of the HCR polymers. Scale bars: 20 μm (in post-expansion units).

FIG. 15: Dependence of RNA FISH spot intensity on degree of expansion and concentration of LabelX. HeLa cells, treated with LabelX diluted to different final concentrations of Label-It Amine concentration, were expanded and stained with a probe-set against GAPDH. After staining, the gelled samples were expanded in 1× PBS (˜2× expansion ratio) and water (˜4× expansion ratio) and the spot intensity for the different samples was quantified. Plotted are mean±standard error; N=6 cells.

DETAILED DESCRIPTION

The present invention provides for the anchoring of nucleic acids into the swellable gel of Expansion Microscopy (ExM), both for in site genomic and transcriptomic assessment, as well as to enable nucleic acid barcodes to be used to identify essentially arbitrary numbers of molecules. International patent application serial number PCT/US15/16788, which is incorporated herein by reference, teaches that the resolution of conventional microscopy can be increased by physically expanding specimens, a process termed ‘expansion microscopy’ (ExM). In short, biological specimens are embedded in a swellable gel material, subjected to a treatment to disrupt native biological networks, and then expanded. The advantages to ExM include tissue clearing, resolution improvement, and higher tolerance to sectioning error due to the specimen expansion in the z-axis.

In ExM, fluorophores were anchored directly to the polymer gel, so that proteins could be visualized; however, RNA molecules were not preserved in the gel and are instead lost during the expansion process. Thus, there was no way to probe the transcriptomic information of the sample.

In one embodiment, the invention provides methods that covalently anchor native nucleic acid molecules and antibody barcodes to the expandable gel matrix of expansion microscopy (ExM). Nucleic acids are modified using a small molecule tag, which lets them participate in free radical polymerization during gelling. During the gel formation step, any biomolecules bearing reactive groups are anchored into the gel and isotropically separated as the gel expands.

In one embodiment, the invention provides a nucleic acid reactive reagent that also carries a chemical group that can get incorporated into the gel. After treatment of samples with this reagent, nucleic adds, including DNA and RNA, are covalently labeled with this reagent. Afterwards, during gel formation, labeled nucleic acids are covalently incorporated into the gel. Using such anchored nucleic acids, the information in the nucleic acid can be used as a barcode, e.g. barcoded antibodies can be used for multiplexed in situ staining for ExM, enabling “arbitrary-color” imaging.

By covalently anchoring the nucleic acids, existing technologies for reading out RNA and DNA can be applied to the expanded context. These strategies include single molecule FISH (Imaging individual mRNA molecules using multiple singly labeled probes. Nature Methods, 2008 Oct; 5(10):877-9), oligo-paint (“Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes.” PNAS 109.52 (2012): 21301-21306) and many other hybridization based readout strategies. Furthermore, the covalent anchoring allows for sequential hybridization, leading to various multiplexing strategies including serial, spectral, and temporal barcoding schemes. The present invention provides methods for labeling and staining with DNA-barcoded primary antibodies, allowing for an arbitrary number of protein tags to be utilized with ExM, This is a key step towards “infinite color” imaging, since previous the expansion microscopy method only enabled 3-color imaging.

In a further embodiment, the invention provides a method for performing sequential hybridizations against nucleic acids covalently incorporated into an ExM gel. Firstly, buffer condition for hybridizing complementary oligonucleotides bearing fluorophores to the nucleic acids in the ExM gel are provided. Second, the ExM gel is re-embedded in a polyacrylamide gel to minimize distortions resulting from changes in buffer. Third, chemical and enzymatic strategies for removing oligonucleotides hybridized to nucleic acids which are covalently anchored to the gel have been developed, which enables re-staining with the same or different oligonucleotides. Chemical strategies include using formamide and high temperatures to de-hybridize oligonucleotides forming duplexes with nucleic acids in the gel. Enzymatic strategies involve using endonucleases that specifically digest the oligonuetides which are hybridized to nucleic acids while leaving the nucleic acids anchored in the gel intact.

In a further embodiment the invention provides for the multiplexed imaging of proteins and transcripts using Expansion Microscopy. First, a strategy to barcode primary antibodies with oligonucleotides by both covalently and non-covalently associating oligonucleotides with their target antibodies has been developed. While covalent attachment schemes involve reacting to amines and sugar chains found on antibodies, non-covalent attachment schemes use secondary Fab fragments conjugated to oligonucleotide barcodes. Second, a set of conditions for performing immunostaining using these oligonucleotide barcoded primary antibodies has been developed. These conditions include unique buffer compositions for minimizing non-specific binding, as well as temperature ranges for obtaining adequate immunostaining. The oligonucleotides which are reacted to these antibodies possess a chemical group that can be incorporated into the ExM gel to gel formation. Therefore, during gel formation, these oligonucleotides are all anchored into the ExM gel while all proteins are degraded. In addition, a strategy for the multiplexed read out of the oligonucleotides and nucleic acids, including RNA and DNA, in the ExM gel using sequential hybridization has been developed. This approach consists of sequentially hybridizing complementary strands bearing fluorophores to each unique oligonucleotide or nucleic acid, one by one, serially. Finally, the set of capabilities offered by out technique enable exponential barcoding schemes demonstrated recently by a few groups. For instance, this approach allows for barcoding nucleic acids via temporal color barcodes or temporal binary barcodes.

One embodiment of a method for in situ genomic and transcriptomic assessment of target nucleic acids present in a biological sample comprises the steps of:

-   -   a) treating the biological sample with a small molecule linker         capable of linking to at least one target nucleic acid and to a         swellable material;     -   b) embedding the biological sample wherein the small molecule         linker is bound to the at least one target nucleic acid in the         biological sample and to the swellable material;     -   c) subjecting the biological sample to a physical disruption         method;     -   d) swelling the swellable material to form an expanded         biological sample;     -   e) providing at least one oligonucleotide complementary to the         at least one target nucleic acid, wherein the at least one         oligonucleotide hybridizes to the at least one target nucleic         acid; and     -   f) genomically or transcriptomically assessing the expanded         biological sample.

In this and other methods, the small molecule linkers are attached to target nucleic acids via a chemical reactive group capable of covalently binding the target nucleic acid. The small molecule linker may be labeled and or the at least one oligonucleotide may be labeled.

In another embodiment, embedding the biological sample in a swellable material may comprise permeating the biological sample with a composition comprising precursors of a swellable polymer and forming a swellable polymer in situ.

In another embodiment, the at least one target nucleic acid is anchored to the swellable material.

In another embodiment, the physical disruption method is an enzymatic digestion.

In another embodiment of the just described method, the target nucleic acids are DNA and/or RNA.

In another embodiment, the expanded biological sample expresses one or more labeled target nucleic acids.

In another embodiment, the expanded sample may be buffered prior to providing at least one oligonucleotide. After buffering, the expanded sample may be re-embedded in a non-swellable material prior to genomically or transcriptically assessing the expanded biological sample. Buffering enables removal of the at least one oligonucleotide through chemical or enzymatic means. For example, formamide and high temperature could be used to chemically remove the at least one oligonucleotide while endonucleases that specifically digest the at least one oligonucleotide could accomplish the same task enzymatically. After buffering, serial or sequential genomic and transcript assessments may be performed on the same expanded sample by repeating the steps of removing the at least one oligonucleotide and providing either the same or different at least one oligonucleotide.

Methods

a. ExM-FISH and ExM FISH-HCR

Secondary antibodies were conjugated to DNA oligo barcodes bearing 5′ acrydite and 3′ amine via the Solulink commercial kit. After primary and secondary antibody staining, samples were gelled, digested, and expanded following ExM procedure. Following expansion, the gelled samples were re-embeded in a 4% polyacrylamide gel by incubating the expanded gel with acrylamide, bis-acrylamide, and radical initiators. To perform in situ hybridization, gelled samples were incubated with fluorescently labeled oligos and excess oligos were subsequently washed out. To perform in situ hybridization with Hybridization Chain Reaction (HCR) signal amplification, gelled samples were incubated with oligo probes bearing a complementary region to the antibody conjugated oligo barcodes and a site for HCR initiation. After washing out excess probes, HCR hairpins were washed in to initiate the amplification.

b. Primary-Fab Antibody Conjugation and Staining

Fab Secondary antibodies were conjugated to DNA oligo barcodes bearing 5′ acrydite and 3′ amine via the Solulink commercial kit. To conjugate IgG primary antibodies with oligo tagged Fabs. Fabs were incubated with primary antibodies along with fluorescently labeled oligonucleotides complementary to the barcodes. Subsequently, excess fabs and oligos were removed using centrifugal spin filters.

Cultured HeLa cells were fixed with 4% formaldehyde. Subsequently, staining antibody mixture was prepared by mixing appropriate purified primary-fab conjugated in a blocking buffer containing dextran sulfate, normal donkey serum, and rabbit gamma globulin. Finally, fixed cells were incubated with the antibody mixture overnight and any excess was washed off

ExFISH: Design and Validation of RNA Anchoring Chemistry

Because of the nature of the reactions occurring during ExM, covalently linking RNAs directly to the ExM gel is necessary. Although transcripts are crosslinked to proteins during fixation, the strong proteolysis of ExM precludes a reliance on proteins for RNA retention (FIGS. 4A, 4B). Thus, covalently securing RNA molecules directly to the ExM gel via a small molecule linker enables the interrogation of these molecules post-expansion. A reagent was synthesized from two building blocks: a molecule containing both an amine as well as an alkylating group that primarily reacts to the N7 of guanine, and a molecule that contains an amine-reactive succinamide ester and a polymerizable acrylamide moiety. Commercially available reagents exist that satisfy each of these two profiles, such as Label-It Amine (MirusBio) and 6-((Acryloyl)amino)hexanoic acid (Acryloyl-X SE, here abbreviated AcX, Life Technologies; all reagents are listed in Table 1). FIG. 1A depicts this molecule, which enables RNA to be covalently functionalized with a free radical polymerizable group, and which will be referred to as LabelX. As shown in FIG. 5E, LabelX does not impede smFISH readout. The original ExM protocol and the use of LabelX allows a procedure wherein a sample could be treated with LabelX to make its RNAs gel-anchorable, followed by gel formation, proteolysis, and osmotic swelling as performed in the original ExM protocol. Once a sample was thus expanded, the RNAs could then be interrogated through FISH (FIG. 1B).

To quantify RNA transcript anchoring yield after expansion, smFISH probes were used, targeting mRNAs of varying copy number (7 targets, with copy number ranging from ˜10 to ˜10,000 per cell, n=59 cells across all 7 targets). smFISH images, taken with probes delivered before (FIG. 1C) and after (FIG. 1D) expansion, to the same cells, showed no loss of transcript detectability with expansion for both low- and high-copy number transcripts (FIG. 1E). The ratio of transcripts detected was near unity at low transcript counts (e.g., in the 10's), however, more transcripts were detected after expansion for highly expressed mRNAs (e.g., in the 1,000's) (FIGS. 9A, 9B, Table 2). This difference arises from the high density of smFISH spots for these targets in the un-expanded state, with the expansion process de-crowding spots that previously were indistinguishable. For example, for smFISH against ACTB, we were able to resolve individual ACTB mRNA puncta post-expansion even within transcriptional foci in the nucleus (FIG. 1C, versus FIG. 1D), which can be dense with mRNA due to transcriptional bursting. Thus, ExFISH is capable of supporting single molecule RNA readout in the expanded state. Since Label-It also reacts to DNA, the ExFISH process enables uniform expansion of the nucleus (FIGS. 7A-C). The isotropy of ExFISH (FIG. 8) was numerically similar to that observed when protein targets were labeled and expanded in the original ExM protocol'. In recent ExM protocols in which proteins are anchored to the same hydrogel as used in ExFISH, with a similar linker“, the distortion is small (a few percent distortion, in cells and tissues). These earlier results, since they were obtained with similar polymer chemistry, serve to bound the ExFISH distortion. The expansion factor is slightly lower than in our original ExM paper (i.e., ˜3.3× versus ˜4×, expansion factors can be found in Figure Legends of this manuscript) due to the salt required to support hybridization of probes.

Nanoscale Imaging of lncRNA with ExFISH

Long non-coding RNAs (lncRNAs) known to serve structural roles in cell biology were imaged. The IncRNA XIST was imaged. Its role in inactivating the X chromosome may depend on initial association with specific chromatin subregions through a process which is still being revealed¹¹. The pre-expansion image (FIG. 1F) shows two bright globular fluorescent regions, presumably corresponding to the X chromosomes of HEK cells undergoing inactivation¹¹⁻¹³, but post-expansion, individual puncta were apparent both within the globular regions as well as nearby (FIG. 1G). ExFISH was used additionally to examine the previously described¹⁴ ring-shaped morphology of ensembles of NEAT1 lncRNAs (FIG. 1H), which has been hypothesized to play an important role in gene expression regulation and nuclear mRNA retention¹⁵. Before expansion, NEAT1 presents in the form of bright, diffraction-limited puncta (FIG. 1H, FIG. 1I), but after expansion, the ring-shaped morphology becomes clear (FIG. 1H, FIG. 1I). Given the complex 3-D structure of the genome¹⁶, mapping lncRNAs may be useful in defining key chromatin regulatory complexes and their spatial configurations.

Super-Resolved, Multiplexed Imaging of RNA with ExFISH

The combination of covalent RNA anchoring to the ExM gel, and the de-crowding of the local environment that results from expansion, could facilitate strategies that have been proposed for multiplexed RNA readout¹⁷⁻¹⁹ based upon sequential hybridization with multiple probe sets. In order to facilitate multiple cycles of FISH, we re-embedded expanded specimens in charge-neutral polyacrylamide. This process allowed expanded gels to be immobilized for multi-round imaging, and additionally stabilized the expanded specimen throughout salt concentration changes in the protocol. Such re-embedded samples exhibited similar expansion factors as non-re-embedded samples (i.e., ˜3×), and were robust to multiple wash-stain cycles as assessed by repeated application of the same probe set (FIG. 2A, FIG. 9A, showing 5 rounds of smFISH staining against GAPDH on cultured cells). This stability was observed even under stringent wash conditions designed to minimize cycle-to-cycle crosstalk (e.g., 100% formamide). Across the 5 rounds, there was no distortion of the locations of individual RNA spots from round to round (FIG. 2B), nor variance in detection efficiency or signal-to-noise ratio (FIGS. 2C, 2D). Having validated the cycle-to-cycle consistency, we next demonstrated the capability of multiplexed ExFISH by applying probes for GAPDH, UBC, NEAT1, USF2, ACTB, and EEF2 in series, enabling 6 individual RNAs to be identified and localized in the same cell (FIG. 2E, FIG. 9B). Thus, serial FISH is applicable to samples expanded after securing RNA to the swellable polymer as here described, making it straightforward to apply probe sets computationally designed to yield more information per FISH cycle, e.g. MERFISH¹⁸⁻²⁰.

3D Nanoscale Imaging of RNA in Mouse Brain Tissue

ExM allows for facile super-resolution imaging of thick 3-D specimens such as brain tissue on conventional microscopy hardware¹. ExFISH was applied to samples of Thy1-YFP mouse brain tissue²¹, using the YFP protein to delineate neural morphology (FIGS. 3A, 3B). Endogenous YFP protein was anchored to the polyacrylate gel via AcX using the proExM protocol⁹, and RNA anchored via LabelX. Since smFISH yields signals too dim to visualize in intact tissues using confocal imaging, the previously described technique of hybridization chain reaction (HCR)⁵ was applied, in particular the next-generation DNA HCR amplifier architecture⁶ (schematic in FIG. 10). In samples containing mouse cortical and hippocampal regions, mRNAs for YFP (FIG. 3C) and glutamic acid decarboxylase 1 Gad1 (FIG. 3D) were easily visualized using a widefield microscope, with YFP mRNA well localized to YFP-fluorescing cells (FIG. 3E), and Gad1 mRNA localized to a population of cells with characteristic arrangement throughout specific layers of the cortex and hippocampus²². Examining brain specimens at high magnification using a confocal spinning disk microscope revealed that individual transcripts could be distinguished due to the physical magnification of ExM (FIG. 3F, with YFP and Gad1 mRNA highlighted), with even highly overexpressed transcripts (e.g., YFP) cleanly resolved into individual puncta (FIG. 3F). When FISH probes were omitted, minimal background HCR amplification was observed (FIGS. 11A-C). Given that ExM enables super-resolution imaging on diffraction limited microscopes, which can be scaled to very fast imaging speeds²³, we used a commercially available lightsheet microscope on a Thy1-YFP brain slice to enable visualization of multiple transcripts, with single molecule precision, throughout a volume of ˜575 μm×575 μm×160 μm thick in just 3 hours (˜6×10¹⁰ voxels in 3 colors; FIGS. 12A-C).

HCR amplifies a target binding event into a bright fluorescent signal (FIG. 10). A stringent method for assessing detection accuracy is to label individual RNAs with different probe sets bearing different colors^(24,25), which shows that 50-80% of mRNAs thus targeted will be doubly labeled, when assessed in cell culture; a 50% co-localization is interpreted as √{square root over (0.5)}˜70% detection efficiency (assuming probe independence); this is a lower bound as it excludes false positives. In order to assess the false positive and negative rates for single molecule visualization in expanded tissues, pairs of probe sets targeting the same transcript with different initiators were delivered. This scheme results in amplified fluorescent signals of two different colors from the same target (FIGS. 13A-B), giving a measure of the hybridization efficiency. Delivering probe sets against a nonexistent transcript also gives a measure of false positive rate. A probe set was delivered against a missense probe (D1g4 reversed, FIG. 3G(i-ii)) as well as a nonexistent transcript (mCherry, Table 3), using Thy1-YFP mouse brain samples, and found a low but nonzero spatial density of dim, yet amplified, puncta (1 per 61 μm³ in unexpanded coordinates, D1g4 reversed; 1 per 48 μm³, mCherry). Essentially zero of these puncta exhibited co-localization (0/1,209 spots, D1g4 reversed; 4/1,540 spots mCherry). In contrast, when a transcript was present (Actb), a large fraction of the puncta exhibited co-localization (an average of 58% of probes in one color co-localized with other color, 15,866/27,504 spots, FIG. 3H(i-ii), Table 3), indicative of a 75% detection efficiency, comparable to the non-amplified single molecule studies described above.

Two-color HCR ExFISH was used against mRNAs to image their position within cellular compartments such as dendritic spines, which require nanoscale resolution for accurate identification or segmentation. The D1g4 mRNA was probed, which encodes the prominent postsynaptic scaffolding protein PSD-95, and which is known to be dendritically enriched⁷. A degree of co-localization (53%, 5,174/9,795 spots) was obtained, suggesting a high detection efficiency, 73% (FIG. 3I). The mRNA was also probed for Camk2a, finding a detection efficiency of 78% (co-localization, 61%, 8,799/14,440 spots, FIGS. 13D-E). Puncta which were co-localized were focused on, thus suppressing false positive errors, and giving a lower-bound on transcript detection (FIG. 13). Focusing on individual dendrites in these expanded samples revealed that individual D1g4 (FIG. 3J(i-ii)) and Camk2a (FIG. 3K(i-ii)) mRNAs could indeed be detected in a sparse subset of dendritic spines as well as fine dendritic processes. To facilitate multiplexed HCR readout, we developed modified HCR hairpins that can be disassembled using toe-hold mediated strand displacement²⁶ (FIGS. 14A-B). These modified HCR amplifiers enable multiple cycles of HCR by disassembling the HCR polymer between subsequent cycles. Given that neurons can have tens of thousands of synapses, and mRNAs can be low copy number, the ability to map mRNAs at synapses throughout neuronal arbors may be useful for a diversity of questions in neuroscience ranging from plasticity to development to degeneration.

Discussion

A novel reagent, easily synthesized from commercial precursors, that enables RNA to be covalently anchored for expansion microscopy is presented. The resulting procedure, ExFISH, enables RNAs to be probed through single-molecule FISH labeling as well as hybridization chain reaction (HCR) amplification. RNA retention before versus after expansion was validated, finding excellent yield, and de-crowding of RNAs for more accurate RNA counts and localization. This enabled visualization, with nanoscale precision and single molecule resolution, RNA structures such as XIST and NEAT1, long non-coding RNAs whose emergent structure has direct implications for their biological roles. The anchoring was robust enough to support serial smFISH, including repeated washing and probe hybridization steps, and multiplexed readout of RNA identity and location, implying that using probes designed according to specific coding strategies¹⁷⁻¹⁹ would support combinatorial multiplexing, in which each additional cycle yields exponentially more transcript information. The covalent anchoring of RNA to the ExM gel may also support enzymatic reactions to be performed in expanded samples—such as reverse transcription, rolling circle amplification (RCA), fluorescent in situ sequencing (FISSEQ)²⁷, and other strategies for transcriptomic readout or SNP detection²⁸, within intact samples.

ExM, being a physical form of magnification, enables nanoscale resolution even on conventional diffraction limited microscopes. Expanding samples makes them transparent and homogeneous in index of refraction, in part because of the volumetric dilution, and in part because of washout of non-anchored components¹. Thus, strategies combining ExM with fast diffraction limited methods like lightsheet microscopy²³ may result in “best of both worlds” performance metrics: the voxel sizes of classical super-resolution methods, but the voxel acquisition rates of increasingly fast diffraction limited microscopes¹. The de-crowding of RNAs enables another key advantage: reducing the effective size of the self-assembled amplification product of HCR, which were applied here, following the protocols of refs.^(5,6), to enable nanoscale resolution visualization of RNA in intact tissues (a paper conducted in parallel has also recently performed single molecule HCR FISH²⁹). An HCR amplicon of size 500 nm in the post-expanded sample would, because of the greater distance between RNAs, have an effective size of 500/3.5=˜150 nm. The lower packing density of amplicons facilitates the imaging of more transcripts per experiment¹⁹ with nanoscale precision. Other methods of achieving brighter signals may be possible. For example, brighter fluorophores such as quantum dots³⁰ or bottlebrush fluorophores³¹ could obviate the need for signal amplification, in principle. The expanded state may enable better delivery of these and other bulky fluorophores into samples. Other amplification strategies may be possible as well, including enzymatic (e.g., RCA²⁸, tyramide amplification²², HRP amplification) as well as nonenzymatic (e.g., branched DNA) methods, although reaction efficiency and diffusion of reagents into the sample must be considered.

ExFISH may find many uses in neuroscience and other biological fields. In the brain, for example, RNA is known to be trafficked to specific synapses as a function of local synaptic activity³² and intron content³³, and locally translated^(7,34,35), and the presence and translation of axonal RNAs remains under investigation³⁶. It is anticipated that, coupled to straightforward multiplexed coding schemes, this method can be used for transcriptomic profiling of neuronal cell-types in situ, as well as for the super-resolved characterization of neuronal connectivity and synaptic organization in intact brain circuits, key for an integrative understanding of the mechanisms underlying neural circuit function and dysfunction. More broadly, visualizing RNAs within cells, and their relationship with RNA processing and trafficking machinery, may reveal new insights throughout biology and medicine.

Method Information

TABLE 1 List of reagents and suppliers Chemical Part Supplies Chemical Name Supplier Number ExM Gel or Sodium Acrylate Sigma 408220 Preparation (purity note:*) Acrylamide Sigma A9099 N,N′- Sigma M7279 Methylenebisacrylamide Ammonium Persulfate Sigma A3678 N,N,N′,N′- Sigma T7024 Tetramethylethylenediamine VA-044 Wako 27776- 21-2 4-Hydroxy-TEMPO Sigma 176141 Hybrid- Dextran Sulfate Sigma D8906-50g ization SSC Thermo AM9765 Buffer Fisher Formamide Thermo AM9342 Fisher Fixation and Paraformaldehyde Electron 15710 Permeabil- Microscopy ization Tissue-prep Buffered Electron 15742-10 10% Formalin Microscopy Triton X-100 Sigma 93426 Ethyl Alcohol Sigma E7023 Glycine Sigma 50046 10x PBS Thermo AM9624 Fisher Protein Proteinase K New P8107S Digestion England

Ethylenediaminetetraacetic Sigma EDS acid Sodium Chloride Sigma S9888 Tris-HCl Life AM9855 HCR Amplification Buffer Molecular N/A Ampli-

fication Tween 20 Sigma P1379 LabelX LABEL-IT ® Amine Mirus Bio MIR 3900

Acryloyl-X, SE Thermo A20770 Fisher LabelX MOPS Sigma M9381-25G Treatment Reembeded DNAse I Sigma 4716728001 Gels Bind-silane Bind-Silane Sigma GE17-1330-

*check for yellow color upon resuspension: that indicates poor quality; solution should be clear (see http://expansionmicroscopy.org)

indicates data missing or illegible when filed

TABLE 2 Mean (Ratio of # spots detected in Decades individual cells after (Transcript ExM, to # spots Standard Sample p- Abundance) detected before ExM) Deviation size (n) Value 10 s 1.082 0.177 14 0.107 100 s 1.105 0.138 29 3.24 × 10⁻

1000 s 1.596 0.562 16 7.09 × 10⁻

indicates data missing or illegible when filed

TABLE 3 Density Total Spot (Co- Count Volume localized (Averaged Hybrid- analyzed Puncta

Tar Across Co

Co

ization (μm

in per ActB 27504 z 15866 0.577 0.76 236749 0.067 Dlg4 9795 5174 0.528 0.727 236749 0.022 Camk2a 14440 8799 0.609

indicates data missing or illegible when filed

TABLE 4 Accession Probe Sequence Initiat or Type YFP B1 1 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATctcgcccttgct B1 caccat YFP B1 2 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATcaccaccccggt B1 gaacag YFP B1 3 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATtccagctcgacc B1 aggatg YFP B1 4 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATtgtggccgttta B1 cgtcgc YFP B1 5 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATctcgccggacac B1 gctgaa YFP B1 6 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATtaggtggcatcg B1 ccctcg YFP B1 7 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATacttcagggtca B1 gcttgc YFP B1 8 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATcttgccggtggt B1 gcagat YFP B1 9 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATgtgggccagggc B1 acgggc YFP B1 10 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATagccgaaggtgg B1 tcacga YFP B1 11 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATggcgaagcactg B1 caggcc YFP B1 12 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATttcatgtggtcg B1 gggtag YFP B1 13 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATacttgaagaagt B1 cgtgct YFP B1 14 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATgtagccttcggg B1 catggc YFP B1 15 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATaagatggtgcgc B1 tcctgg YFP B1 16 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATagttgccgtcgt B1 ccttga YFP B1 17 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATcacctcggcgcg B1 ggtctt YFP B1 18 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATagggtgtcgccc B1 tcgaac YFP B1 19 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATtcagctcgatgc B1 ggttca YFP B1 20 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATctccttgaagtc B1 gatgcc YFP B1 21 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATtgccccaggatg B1 ttgccg YFP B1 22 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATtgtagttgtact B1 ccagct YFP B1 23 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATgatatagacgtt B1 gtggct YFP B1 24 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATttcttctgcttg B1 tcggcc YFP B1 25 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATtgaagttcacct B1 tgatgc YFP B1 26 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATctcgatgttgtg B1 gcggat YFP B1 27 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATgcgagctgcacg B1 ctgccg YFP B1 28 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATtgttctgctggt B1 agtggt YFP B1 29 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATggggccgtcgcc B1 gatggg YFP B1 30 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATtggttgtcgggc B1 agcagc YFP B1 31 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATcggactggtagc B1 tcaggt YFP B1 32 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATgttggggtcttt B1 gctcag YFP B1 33 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATaccatgtgatcg B1 cgcttc YFP B1 34 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATcggtcacgaact B1 ccagca YFP B1 35 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATgccgagagtgat B1 cccggc YFP B1 36 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTAATtacttgtacagc B1 tcgtcc Gad1 21 TTGAAAAATCGAGGGTGACCTGAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 CAATCATC Gad1 22 CCAATGATATCCAAACCAGTAGAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 gggAAgAg Gad1 23 GATGTCAGCCATTCACCAGCTAAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 CAATCATC Gad1 24 TCATATGTGAACATATTGGTATAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 gggAAgAg Gad1 25 ATGAGAACAAACACGGGTGCAAAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 CAATCATC Gad1 26 TCTCTCATCTTCTTAAGAGTAAAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 gggAAgAg Gad1 27 TCTTTATTTGACCATCCAACGAAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 CAATCATC Gad1 28 GCTCCCCCAGGAGAAAATATCCAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 gggAAgAg Gad1 29 ATGATGCTGTACATATTGGATAAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 CAATCATC Gad1 30 ACTTCTGGGAAGTACTTGTAACAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 gggAAgAg Gad1 31 ACAGCCGCCATGCCTTTTGTCTAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 CAATCATC Gad1 32 TGTTCTGAGGTGAAGAGGACCAAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 gggAAgAg Gad1 33 GCTTTCTTTATGGAATAGTGACAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 CAATCATC Gad1 34 TTGTCGGTTCCAAAGCCAAGCGAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 gggAAgAg Gad1 35 TCATTGCACTTTATCAAAATCAAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 CAATCATC Gad1 36 TCTAAATCAGCCGGAATTATCTAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 gggAAgAg Gad1 37 TGTTTGGCATCAAGAATTTTTGAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 CAATCATC Gad1 38 GCATTGACATAAAGGGGAACATAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 gggAAgAg Gad1 39 CCGTAAACAGTCGTGCCTGCGGAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 CAATCATC Gad1 40 TCCGCAATTTCCTGGATTGGATAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 gggAAgAg Gad1 41 CAAAGGTTGTATTTCTCACATAAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 CAATCATC Gad1 42 CCACCACCCCAGGCAGCATCCAAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 gggAAgAg Gad1 43 CGGTGCTTCCGGGACATGAGCAAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 CAATCATC Gad1 44 TTGGCCCTTTCTATGCCGCTGAAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 gggAAgAg Gad1 45 TTGTGAGGGTTCCAGGTGACTGAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 CAATCATC Gad1 46 GCAGAGCACTGGAGCAGCACGCAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 gggAAgAg Gad1 47 ATACCCTTTTCCTTGACCAGAAAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 CAATCATC Gad1 48 CCTGCACACATCTGGTTGCATCAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 gggAAgAg ActB B2 2 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAAggaatacagcccgg B2 ggagcatc ActB B2 4 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAAcacccacataggag B2 tccttctg ActB B2 6 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAAcaatggggtacttc B2 agggtcag ActB B2 8 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAAggtgccagatcttc B2 tccatgtc ActB B2 10 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAAtcatcttttcacgg B2 ttggcctt ActB B2 12 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAAtggctacgtacatg B2 gctggggt ActB B2 14 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAAcaatgcctgtggta B2 cgaccaga ActB B2 16 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAAcctcgtagatgggc B2 acagtgtg ActB B2 18 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAAatcttcatgaggta B2 gtctgtca ActB B2 20 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAAatttccctctcagc B2 tgtggtgg ActB B2 22 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAAtcgaagtctagagc B2 aacatagc ActB B2 24 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAAtagctcttctccag B2 ggaggaag ActB B2 26 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAAcggaaccgctcgtt B2 gccaatag ActB B2 28 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAAcaggattccatacc B2 caagaagg ActB B2 30 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAAtcaacgtcacactt B2 catgatgg ActB B2 32 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAAgtggtaccaccaga B2 cagcactg ActB B2 34 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAAagagcagtaatctc B2 cttctgca ActB B2 36 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAAttgcgctcaggagg B2 agcaatga ActB B2 38 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAAaaggtggacagtga B2 ggccagga ActB B2 40 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAAgaggggccggactc B2 atcgtact Act Short gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTTgcgcagcgatatcg B1 HCR 1 tcatccat Act Short gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTTccattcccaccatc B1 HCR 3 acaccctg Act Short gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTTtacctctcttgctc B1 HCR 5 tgggcctc Act Short gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTTcccagttggtaaca B1 HCR 7 atgccatg Act Short gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTTcacgcagctcattg B1 HCR 9 tagaaggt Act Short gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTTtgaaggtctcaaac B1 HCR 11 atgatctg Act Short gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTTcatacagggacagc B1 HCR 13 acagcctg Act Short gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTTtgaccccgtctccg B1 HCR 15 gagtccat Act Short gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTTggatggcgtgaggg B1 HCR 17 agagcata Act Short gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTTaagctgtagccacg B1 HCR 19 ctcggtca Act Short gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTTagcttctctttgat B1 HCR 21 gtcacgca Act Short gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTTgatgcggcagtggc B1 HCR 23 catctcct Act Short gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTTatgacctggccgtc B1 HCR 25 aggcagct Act Short gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTTggctggaaaagagc B1 HCR 27 ctcagggc Act Short gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTTttgaatgtagtttc B1 HCR 29 atggatgc Act Short gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTTttggcatagaggtc B1 HCR 31 tttacgga Act Short gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTTctgtcagcaatgcc B1 HCR 33 tgggtaca Act Short gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTTttgatcttcatggt B1 HCR 35 gctaggag Act Short gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTTgagccaccgatcca B1 HCR 37 cacagagt Act Short gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTTtgcttgctgatcca B1 HCR 39 catctgct Act Short gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTTtagaagcacttgcg B1 HCR 41 gtgcacga Act Short gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgTTtagaagcacttgcg B1 HCR 41 gtgcacga DLG4 B1 2 GGGCTGTGTTCCAGAGGGGGCGAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 gggAAgAg DLG4 B1 4 GTGTCCGTGTTGACAATCACAGAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 gggAAgAg DLG4 B1 6 TCCTCATACTCCATCTCCCCCTAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 gggAAgAg DLG4 B1 8 GTGCCACCTGCGATGCTGAAGCAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 gggAAgAg DLG4 B1 10 GGAATGATCTTGGTGATAAAGAAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 gggAAgAg DLG4 B1 12 AACAGGATGCTGTCGTTGACCCAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 gggAAgAg DLG4 B1 14 AGGGCCTCCACTGCAGCTGAATAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 gggAAgAg DLG4 B1 16 GCTGGGGGTTTCCGGCGCATGAAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 gggAAgAg DLG4 B1 18 CTGAAGCCAAGTCCTTTAGGCCAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 gggAAgAg DLG4 B1 20 ACGTAGATGCTATTATCTCCAGAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 gggAAgAg DLG4 B1 22 CCGATCTGCAACCTGCCATCCTAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 gggAAgAg DLG4 B1 24 TCCTCATGCATGACATCCTCTAAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 gggAAgAg DLG4 B1 26 TTGGCCACCTTTAGGTACACAAAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 gggAAgAg DLG4 B1 28 GAGGTTGTGATGTCTGGGGGAGAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 gggAAgAg DLG4 B1 30 TCGGTGCCCAAGTAGCTGCTATAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 gggAAgAg DLG4 B2 1 TCTTCATCTTGGTAGCGGTATTAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 CAATCATC DLG4 B2 3 GGAGAATTGGCCTGGTTGGGGAAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 CAATCATC DLG4 B2 5 GTTCCGTTCACATATCCTGGGGAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 CAATCATC DLG4 B2 7 AGACCTGAGTTACCCCTTTCCAAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 CAATCATC DLG4 B2 9 GATGGGTCGTCACCGATGTGTGAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 CAATCATC DLG4 B2 11 AGGCGGCCATCCTGGGCTGCAGAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 CAATCATC DLG4 B2 13 GTCACCTCCCGGACATCCACTTAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 CAATCATC DLG4 B2 15 TAGAGGCGAACGATGGAACCCGAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 CAATCATC DLG4 B2 17 TTGATAAGCTTGATCTCTATGAAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 CAATCATC DLG4 B2 19 ATGTGCTGGTTCCCAACGCCCCAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 CAATCATC DLG4 B2 21 TGGGCAGCGCCTCCTTCGATGAAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 CAATCATC DLG4 B2 23 CCCACACTGTTGACCGCCAGGAAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 CAATCATC DLG4 B2 25 TCATATGTGTTCTTCAGGGCTGAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 CAATCATC DLG4 B2 27 TAGCTGTCACTCAGGTAGGCATAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 CAATCATC DLG4 B2 29 CTGATCTCATTGTCCAGGTGCTAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 CAATCATC Camk2a iso2 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgAACGGGTGCAGGTGAT B1 1 GGTAGCCA Camk2a iso2 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAATCCTCAAAGAGCTG B2 2 GTACTCTT Camk2a iso2 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgAAACAGAGAAGGCTCC B1 3 CTTTCCCA Camk2a iso2 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAACCAGCCAGCACCTT B2 4 CACACACC Camk2a iso2 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgAAATAATCTTGGCAGC B1 5 ATACTCCT Camk2a iso2 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAATGATCTCTGGCTGA B2 6 AAGCTTCT Camk2a iso2 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgAACGGGCCTCACGCTC B1 7 CAGCTTCT Camk2a iso2 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAAATATTGGGGTGCTT B2 8 CAACAAGC Camk2a iso2 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgAAGAGATGCTGTCATG B1 9 GAGTCGGA Camk2a iso2 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAATCGAAGATAAGGTA B2 10 GTGGTGCC Camk2a iso2 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgAAAACAGTTCCCCACC B1 11 AGTAACCA Camk2a iso2 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAACTGTAATACTCCCG B2 12 GGCCACAA Camk2a iso2 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgAAATACAGTGGCTGGC B1 13 ATCAGCTT Camk2a iso2 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAACAGTGTAGCACAGC B2 14 CTCCAAGA Camk2a iso2 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgAACGATGCACCACCCC B1 15 CATCTGGT Camk2a iso2 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAAGCCAGCAACAGATT B2 16 CTCAGGCT Camk2a iso2 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgAAACAGCAGCGCCCTT B1 17 GAGCTTCG Camk2a iso2 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAATCTATGGCCAGGCC B2 18 AAAGTCTG Camk2a iso2 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgAACATGCCTGCTGCTC B1 19 CCCCTCCA Camk2a iso2 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAAAGGTATCCAGGTGT B2 20 CCCTGCGA Camk2a iso2 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgAATCCTTCCTCAGCAC B1 21 TTCTGGGG Camk2a iso2 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAAGCCCACAGGTCCAC B2 22 GGGCTTCC Camk2a iso2 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgAAAAGATATACAGGAT B1 23 GACGCCAC Camk2a iso2 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAATCATCCCAGAACGG B2 24 GGGATACC Camk2a iso2 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgAATGCTGGTACAGGCG B1 25 ATGCTGGT Camk2a iso2 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAAGATGGGAAATCATA B2 26 GGCACCAG Camk2a iso2 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgAAGGGGTGACGGTGTC B1 27 CCATTCTG Camk2a iso2 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAAAGCATCTTATTGAT B2 28 CAGATCCT Camk2a iso2 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgAAATGCGTTTGGACGG B1 29 GTTGATGG Camk2a iso2 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAACATGGGTGCTTGAG B2 30 AGCCTCAG Camk2a iso2 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgAAGCCACGGTGGAGCG B1 31 GTGCGAGA Camk2a iso2 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAATCCACGGTCTCCTG B2 32 TCTGTGCA Camk2a iso2 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgAACTGGCATTGAACTT B1 33 CTTCAGGC Camk2a iso2 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAAGTGGTGAGGATGGC B2 34 TCCCTTCA Camk2a iso2 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgAAGAGAAGTTCCTGGT B1 35 GGCCAGCA Camk2a iso2 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAATTCTTCTTGTTTCC B2 36 TCCGCTCT Camk2a iso2 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgAATCAGAAGATTCCTT B1 37 CACACCAT Camk2a iso2 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAATCTTCGTCCTCAAT B2 38 GGTGGTGT Camk2a iso2 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgAAATTTCCTGTTTGCG B1 39 CACTTTGG Camk2a iso2 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAAGCTTCGATCAGCTG B2 40 CTCTGTCA Camk2a iso2 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgAAGACTCAAAGTCTCC B1 41 ATTGCTTA Camk2a iso2 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAAGTCATTCCAGGGTC B2 42 GCACATCT Camk2a iso2 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgAACCCAGGGCCTCTGG B1 43 TTCAAAGG Camk2a iso2 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAACGATGAAAGTCCAG B2 44 GCCCTCCA Camk2a iso2 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgAAGACCACAGGTTTTC B1 45 AAAATAGA Camk2a iso2 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAAATGGTGGTGTGCAC B2 46 GGGCTTGC Camk2a iso2 gAggAgggCAgCAAACgggAAgAgTCTTCCTTTACgAAATCAGGTGGATGTG B1 47 AGGGTTCA Camk2a iso2 CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCAAATATAGGCGATGCA B2 48 GGCTGACT mCherry 2C cttcttcaccttttgaaaccatAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 1 gggAAgAg mCherry 2C ccatatgaactttaaatctcatAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 3 gggAAgAg mCherry 2C cttcaccttcaccttcaatttcAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 5 gggAAgAg mCherry 2C cacctttagtaactttcaatttAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 7 gggAAgAg mCherry 2C catacataaattgtggtgacaaAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 9 gggAAgAg mCherry 2C ttaaataatctggaatatcagcAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 11 gggAAgAg mCherry 2C tcaaaattcataactctttcccAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 13 gggAAgAg mCherry 2C ctctcaatttaactttataaatAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 15 gggAAgAg mCherry 2C ccatagtttttttttgcataacAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 17 gggAAgAg mCherry 2C tcaatctttgtttaatttcaccAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 19 gggAAgAg mCherry 2C taatattaacattataagcaccAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 21 gggAAgAg mCherry 2C tttcatattgttcaacaatagtAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 23 gggAAgAg mCherry 2C attctttaataatagccatattAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 2 CAATCATC mCherry 2C attcatgaccattaactgaaccAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 4 CAATCATC mCherry 2C cagtttgagtaccttcatatggAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 6 CAATCATC mCherry 2C tatcccaagcaaatggtaatggAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 8 CAATCATC mCherry 2C gatgtttaacataagcttttgaAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 10 CAATCATC mCherry 2C ttaaaaccttctggaaatgacaAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 12 CAATCATC mCherry 2C gagtaacagtaacaacaccaccAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 14 CAATCATC mCherry 2C gaccatctgatggaaaattagtAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 16 CAATCATC mCherry 2C ttctttctgatgaagcttcccaAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 18 CAATCATC mCherry 2C gtaattgaactggttttttagcAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 20 CAATCATC mCherry 2C tcattatgtgaagtaatatccaAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 22 CAATCATC mCherry 2C atttatataattcatccataccAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 24 CAATCATC DLG4 AATACCGCTACCAAGATGAAGAAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 ShHCR mis 1 CAATCATC DLG4 TCCCCAACCAGGCCAATTCTCCAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 ShHCR mis 3 CAATCATC DLG4 CCCCAGGATATGTGAACGGAACAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 ShHCR mis 5 CAATCATC DLG4 TGGAAAGGGGTAACTCAGGTCTAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 ShHCR mis 7 CAATCATC DLG4 CACACATCGGTGACGACCCATCAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 ShHCR mis 9 CAATCATC DLG4 CTGCAGCCCAGGATGGCCGCCTAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 ShHCR mis 11 CAATCATC DLG4 AAGTGGATGTCCGGGAGGTGACAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 ShHCR mis 13 CAATCATC DLG4 CGGGTTCCATCGTTCGCCTCTAAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 ShHCR mis 15 CAATCATC DLG4 TCATAGAGATCAAGCTTATCAAAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 ShHCR mis 17 CAATCATC DLG4 GGGGCGTTGGGAACCAGCACATAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 ShHCR mis 19 CAATCATC DLG4 TCATCGAAGGAGGCGCTGCCCAAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 ShHCR mis 21 CAATCATC DLG4 TCCTGGCGGTCAACAGTGTGGGAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 ShHCR mis 23 CAATCATC DLG4 CAGCCCTGAAGAACACATATGAAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 ShHCR mis 25 CAATCATC DLG4 ATGCCTACCTGAGTGACAGCTAAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 ShHCR mis 27 CAATCATC DLG4 AGCACCTGGACAATGAGATCAGAAAgCTCAgTCCATCCTCgTAAATCCTCAT B2 ShHCR mis 29 CAATCATC DLG4 CGCCCCCTCTGGAACACAGCCCAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 ShHCR mis 2 gggAAgAg DLG4 CTGTGATTGTCAACACGGACACAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 ShHCR mis 4 gggAAgAg DLG4 AGGGGGAGATGGAGTATGAGGAAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 ShHCR mis 6 gggAAgAg DLG4 GCTTCAGCATCGCAGGTGGCACAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 ShHCR mis 8 gggAAgAg DLG4 TCTTTATCACCAAGATCATTCCAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 ShHCR mis 10 gggAAgAg DLG4 GGGTCAACGACAGCATCCTGTTAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 ShHCR mis 12 gggAAgAg DLG4 ATTCAGCTGCAGTGGAGGCCCTAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 ShHCR mis 14 gggAAgAg DLG4 TCATGCGCCGGAAACCCCCAGCAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 ShHCR mis 16 gggAAgAg DLG4 GGCCTAAAGGACTTGGCTTCAGAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 ShHCR mis 18 gggAAgAg DLG4 CTGGAGATAATAGCATCTACGTAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 ShHCR mis 20 gggAAgAg DLG4 AGGATGGCAGGTTGCAGATCGGAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 ShHCR mis 22 gggAAgAg DLG4 TAGAGGATGTCATGCATGAGGAAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 ShHCR mis 24 gggAAgAg DLG4 TTGTGTACCTAAAGGTGGCCAAAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 ShHCR mis 26 gggAAgAg DLG4 CTCCCCCAGACATCACAACCTCAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 ShHCR mis 28 gggAAgAg DLG4 ATAGCAGCTACTTGGGCACCGAAAgCATTCTTTCTTgAggAgggCAgCAAAC B1 ShHCR mis 30 gggAAgAg smFISH Probe Name Oligonucleotide Sequence Sequence Name UBC atggtcttaccagtcagagt hUBC_1 gacattctcgatggtgtcac hUBC_2 gggatgccttccttatcttg hUBC_3 atcttccagctgttttccag hUBC_4 cagtgagtgtcttcacgaag hUBC_5 tcctggatctttgctttgac hUBC_6 cagggtagactctttctgga hUBC_7 cttcacgaagatctgcatcc hUBC_8 tcttggatctttgccttgac hUBC_9 cagtgagtgtcttcacgaag hUBC_10 tgacgttctcgatagtgtca hUBC_11 tccttgtcttggatctttgc hUBC_12 cagggtagactctttctgga hUBC_13 cttcacgaagatctgcatcc hUBC_14 agagtgatggtcttaccagt hUBC_15 tcttggatctttgccttgac hUBC_16 cttcacgaagatctgcatcc hUBC_17 agagtgatggtcttaccagt hUBC_18 tcttggatctttgccttgac hUBC_19 tgtttcccagcaaagatcaa hUBC_20 cttcacgaagatctgcatcc hUBC_21 agagtgatggtcttaccagt hUBC_22 tcttggatctttgccttgac hUBC_23 tgtttcccagcaaagatcaa hUBC_24 cttcacgaagatctgcatcc hUBC_25 agagtgatggtcttaccagt hUBC_26 tcttggatctttgccttgac hUBC_27 tgtttcccagcaaagatcaa hUBC_28 gacattctcgatggtgtcac hUBC_29 gggatgccttccttatcttg hUBC_30 tgtttcccagcaaagatcaa hUBC_31 agagtggactctttctggat hUBC_32 EEF2 atctggtctaccgtgaagtt hEEF2_1 ttggccttcttgtccatgat hEEF2_2 gtatcagtgaagcgtgtctc hEEF2_3 ttgacttgatggtgatgcaa hEEF2_4 ctcgtagaagagggagatgg hEEF2_5 tccttgctctgcttgatgaa hEEF2_6 gggagtcaatgaggttgatg hEEF2_7 cggtccatcttgttcatcat hEEF2_8 gtggagatgatgacgttcac hEEF2_9 gtaccgaggacaggatcgat hEEF2_10 caaactgcttcagggtgaag hEEF2_11 aacttggccacatacatctc hEEF2_12 atgtcctctactttcttggc hEEF2_13 ttcatgatcgcatcaaacac hEEF2_14 gtccagtttgatgtccagtt hEEF2_15 gatggtgatcatctgcaaca hEEF2_16 tttggggtcacagcttttaa hEEF2_17 gtagaaccgacctttgtcgg hEEF2_18 ccatgatcctgaccttcagg hEEF2_19 ttcttcccaggggtatagtt hEEF2_20 tctggattggcttcaggtag hEEF2_21 ggcccatcatcaagattgtt hEEF2_22 gtcttcaccaggaactggtc hEEF2_23 ctgacgctgaacttcatcac hEEF2_24 atgatatgctctcccgactc hEEF2_25 gactcttcactgaccgtctc hEEF2_26 cttcatgtacagccggttgt hEEF2_27 tcgcctttatcgatgtcctc hEEF2_28 tgatgtcggtgaggatgttg hEEF2_29 cactgtccttgatctcgttg hEEF2_30 gtcagcacactggcatagag hEEF2_31 atctccacaaggtagatggg hEEF2_32 USF2 ggatccagacccgggtccag usf2_withUT R_1 tactggatgttgtggtcgcc usf2_withUT R_2 catttgtctctgtgcggaac usf2_withUT R_3 attttggatcacagcctgtc usf2_withUT R_4 gactgccaccattgctgaag usf2_withUT R_5 ctgggaaataggcaaatcgt usf2_withUT R_6 gacacagccgtagtatctcc usf2_withUT R_7 gtctgaagcacatcctgggg usf2_withUT R_8 ggcgatcgtcctctgtgttc usf2_withUT R_9 tggttccatcaatttttgga usf2_withUT R_10 ttctcctctcatctcggggt usf2_withUT R_11 ctccacttcgttgtgctggg usf2_withUT R_12 cagttgttgatcttgtccct usf2_withUT R_13 gattttcgaaagctggacga usf2_withUT R_14 gttgtctgcgttacagtctg usf2_withUT R_15 ggccttggacaggatccctc usf2_withUT R_16 cgcaactcccggatgtaatc usf2_withUT R_17 ctgcatgcgctggttggtct usf2_withUT R_18 gctcggcctctttgaaggtc usf2_withUT R_19 agctcgttgtccatctgcag usf2_withUT R_20 caccatctccaggttgtgct usf2_withUT R_21 tgtatccacagaaatgcatt usf2_withUT R_22 ggaggataccgtttccaagt usf2_withUT R_23 gtgagaccactagaagtgcc usf2_withUT R_24 cataggtccaggccccgggt usf2_withUT R_25 cagggacccagaaacaagag usf2_withUT R_26 gggccagtttattgcagtta usf2_withUT R_27 TOP2A ctgggcggagcaaaatatgt hTOP2A_CD S_1 tcttcatcgtaaacccacat hTOP2A_CD S_2 ccggatcaattgtgactcta hTOP2A_CD S_3 ccttttccattattccatat hTOP2A_CD S_4 agaagttaggagctgtccaa hTOP2A_CD S_5 ccagcaatatcatatgctct hTOP2A_CD S_6 ttactggcagtttatttcca hTOP2A_CD S_7 tgttgatccaaagctcttgg hTOP2A_CD S_8 aactggacttgggccttaaa hTOP2A_CD S_9 atcattggcatcatcgagtt hTOP2A_CD S_10 gtcaggataagcgtacactc hTOP2A_CD S_11 ggaaaaccccatatttgtct hTOP2A_CD S_12 tttcttgtactgaagaccca hTOP2A_CD S_13 ttggtcctgatctgtcataa hTOP2A_CD S_14 ctccagaaaacgatgtcgca hTOP2A_CD S_15 gttaaccattcctttcgatc hTOP2A_CD S_16 agctaattgggcaaccttta hTOP2A_CD S_17 atgtatcgtggactagcaga hTOP2A_CD S_18 acgctggttgtcatcatata hTOP2A_CD S_19 ttcttctccatccatcaaac hTOP2A_CD S_20 cccttgaagttcttgtaact hTOP2A_CD S_21 tatgagaggaggtgtcttct hTOP2A_CD S_22 tgtatggtattccctatagt hTOP2A_CD S_23 tcagtttagcagattcagca hTOP2A_CD S_24 cttcacaggatccgaatcat hTOP2A_CD S_25 gtggaatgactctttgacca hTOP2A_CD S_26 tgctcctatctgattctgaa hTOP2A_CD S_27 agtggaggtggaagactgac hTOP2A_CD S_28 aattcaaagctggatccctt hTOP2A_CD S_29 caggatcaggcttttgagag hTOP2A_CD S_30 cttggatttcttgcttgtga hTOP2A_CD S_31 tatggaagtcatcactctcc hTOP2A_CD S_32 NEAT1 gacctagtctccttgccaag NEAT1_1 ggatattttccatgcagcct NEAT1_2 acaagttgaagattagccct NEAT1_3 ccttggtctggaaaaaaagg NEAT1_4 cgagctaagttcagttccac NEAT1_5 ggccgagcgaaaattacata NEAT1_6 cctgtcaaacatgctaggtg NEAT1_7 actgccacctggaaaataaa NEAT1_8 gtgagctcacaagaagagtt NEAT1_9 accagatgaccaggtaatgt NEAT1_10 cggtccatgaagcatttttg NEAT1_11 tcgccatgaggaacactata NEAT1_12 aatctgcaggcatcaattga NEAT1_13 cctggaaacagaacattgga NEAT1_14 gcatctgctgtggacttttt NEAT1_15 ggctctggaacaagcattta NEAT1_16 tgcagcatctgaaaaccttt NEAT1_17 accggaggctcaatttagaa NEAT1_18 caaggttccaagcacaaaac NEAT1_19 acagcttagggatcttcttg NEAT1_20 tggcatcaacgttaaaatgt NEAT1_21 tctacaaggcatcaatctgc NEAT1_22 aagaacttctccgagaaacg NEAT1_23 gccccaagttatttcatcag NEAT1_24 gcgtttagcacaacacaatg NEAT1_25 ggaatgaccaacttgtaccc NEAT1_26 caatgcccaaactagacctg NEAT1_27 tcctagtaatctgcaatgca NEAT1_28 agcaagaacaaaagagcact NEAT1_29 ggtcctcttactagaatgcc NEAT1_30 ctgtgtcacctgttttcagt NEAT1_31 cctttggttctcggaaaact NEAT1_32 agctggtaaagacatttccc NEAT1_33 ctctgaaacaggctgtcttg NEAT1_34 gcccatctttcaagtgacta NEAT1_35 aaccacctaagttgctaagg NEAT1_36 tcgtcttaagtggtccctta NEAT1_37 atccagaagagcccatctaa NEAT1_38 acctgtgacaaatgaggaac NEAT1_39 agatgtgtttctaaggcacg NEAT1_40 acagtgaccacaaaaggtta NEAT1_41 agcaaaggtacatggattct NEAT1_42 cagggttttcagatcacaca NEAT1_43 ccccaagtcattggttaaga NEAT1_44 tcccaacgacagtaattgtt NEAT1_45 cccatacatgcgtgactaat NEAT1_46 caacagcatacccgagacta NEAT1_47 acagagcaacataccagtac NEAT1_48

Cell Culture and Fixation

HeLa (ATCC CCL-2) cells and HEK293-FT cells (Invitrogen) were cultured on Nunc Lab-Tek II Chambered Coverglass (Thermo Scientific) in D10 medium (Cellgro) supplemented with 10% FBS (Invitrogen), 1% penicillin/streptomycin (Cellgro), and 1% sodium pyruvate (BioWhittaker). Cells were authenticated by the manufacturer and tested for mycoplasma contamination to their standard levels of stringency, and were here used because they are common cell lines for testing new tools. Cultured cells were washed once with DPBS (Cellgro), fixed with 10% formalin for 10 mins, and washed twice with 1× PBS. Fixed cells were then stored in 70% Ethanol at 4° C. until use.

Preparation of LabelX

Acryloyl-X, SE (6-((acryloyl)amino)hexanoic acid, succinimidyl ester, here abbreviated AcX; Thermo-Fisher) was resuspended in anhydrous DMSO at a concentration of 10 mg/mL, aliquoted and stored frozen in a desiccated environment. LABEL-IT® Amine Modifying Reagent (Mirus Bio, LLC) was resuspended in the provided Mirus Reconstitution Solution at 1 mg/ml and stored frozen in a desiccated environment. To prepare LabelX, 10 μL, of AcX (10 mg/mL) was reacted with 100 μL of LABEL-IT® Amine Modifying Reagent (1 mg/mL) overnight at room temperature with shaking. LabelX was subsequently stored frozen (−20° C.) in a desiccated environment until use.

Mouse perfusion

All methods for animal care and use were approved by the Massachusetts Institute of Technology Committee on Animal Care and were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All solutions below were made up in 1× phosphate buffered saline (PBS) prepared from nuclease free reagents. Mice were anesthetized with isoflurane and perfused transcardially with ice cold 4% paraformaldehyde. Brains were dissected out, left in 4% paraformaldehyde at 4° C. for one day, before moving to PBS containing 100 mM glycine. Slices (50 μm and 200 μm) were sliced on a vibratome (Leica VT1000S) and stored at 4° C. in PBS until use. The mouse used in FIG. 3 and related analyses was a Thy1-YFP (Tg(Thy1-YFP)16 Jrs) male mouse in the age range 6-8 weeks. No sample size estimate was performed, since the goal was to demonstrate a technology. No exclusion, randomization or blinding of samples was performed.

LabelX Treatment of Cultured Cells and Brain Slices

Fixed cells were washed twice with 1× PBS, once with 20 mM MOPS pH 7.7, and incubated with LabelX diluted to a desired final concentration in MOPS buffer (20 mM MOPS pH 7.7) at 37° C. overnight followed by two washes with 1× PBS. For cells, ranges of LabelX were used that resulted in a LABEL-IT® Amine concentration of 0.006-0.02 mg/mL; higher concentrations resulted in somewhat dimmer smFISH staining (FIG. 15), but otherwise no difference in staining quality was observed with LABEL-IT® Amine concentrations in this range. For FIG. 1 e, FIG. 4, FIG. 5, and FIG. 6 fixed cells were incubated with LabelX diluted to a final LABEL-IT® Amine concentration of 0.02 mg/mL. For all other experiments in cells, fixed cells were treated with LabelX diluted to a final LABEL-IT® Amine concentration of 0.006 mg/mL.

Brain slices, as prepared above, were incubated with 20 mM MOPS pH 7.7 for 30 mins and subsequently incubated with LabelX diluted to a final LABEL-IT® Amine concentration of 0.1 mg/mL (due to their increased thickness and increased fragmentation from formaldehyde post-fixation) in MOPS buffer (20 mM MOPS pH 7.7) at 37° C. overnight. For YFP retention, slices were treated with 0.05 mg/mL AcX in PBS for >6 hours @ RT.

smFISH in Fixed Cultured Cells Before Expansion

Fixed cells were briefly washed once with wash buffer (10% formamide, 2× SSC) and hybridized with RNA FISH probes in hybridization buffer (10% formamide, 10% dextran sulfate, 2× SSC) overnight at 37° C. Following hybridization, samples were washed twice with wash buffer, 30 mins per wash, and washed once with 1× PBS. Imaging was performed in 1× PBS.

smFISH probe sets targeting the human transcripts for TFRC, ACTB, GAPDH, XIST, and 5′ portion of NEAT1 were ordered from Stellaris with Quasar 570 dye. Probe sets against UBC, EEF2, USF2, TOP2A and full length NEAT1 were synthesized, conjugated to fluorophores, and subsequently purified by HPLC as described previously³⁷. Oligonucleotide sequences for probe sets and accession numbers can be found in Table 4.

Gelation, Digestion and Expansion

Monomer solution (1× PBS, 2 M NaCl, 8.625% (w/w) sodium acrylate, 2.5% (w/w) acrylamide, 0.15% (w/w) N,N′-methylenebisacrylamide) was mixed, frozen in aliquots, and thawed before use. Monomer solution was cooled to 4° C. before use. For gelling cultured cells treated with LabelX, a concentrated stock of VA-044 (25% w/w, chosen instead of the Ammonium persulfate (APS)/Tetramethylethylenediamine (TEMED) of the original ExM protocol¹ because APS/TEMED resulted in autofluorescence that was small in magnitude but appreciable in the context of smFISH) was added to the monomer solution to a final concentration of 0.5% (w/w) and degassed in 200 μl aliquots for 15 mins. Cells were briefly incubated with the monomer solution plus VA-044 and transferred to a humidified chamber. Subsequently, the humidified chamber was purged with nitrogen gas. To initiate gelation, the humidified chamber was transferred to a 60° C. incubator for two hours. For gelling brain slices treated with LabelX, gelation was performed as in the original ExM protocol (since, with HCR amplification, the slight autofluorescence of APS/TEMED was negligible). Gelled cultured cells and brain slices were digested with Proteinase K (New England Biolabs) diluted 1:100 to 8 units/mL in digestion buffer (50 mM Tris (pH 8), 1 mM EDTA, 0.5% Triton X-100, 500 mM NaCl) and digestion was carried out overnight at 37° C. The gels expand slightly in the high osmolarity digestion buffer (˜1.5×). After digestion, gels were stored in 1× PBS until use and expansion was carried out as previously described.

smFISH Staining After Expansion

Expanded gels were incubated with wash buffer (10% formamide, 2× SSC) for 30 mins at room temperature and hybridized with RNA FISH probes in hybridization buffer (10% formamide, 10% dextran sulfate, 2× SSC) overnight at 37° C. Following hybridization, samples were washed twice with wash buffer, 30 minutes per wash, and washed once with1× PBS for another 30 mins. Imaging was performed in 1× PBS.

Image Processing and Analysis of smFISH performed on Cultured Cells

Widefield images of smFISH staining performed before or after expansion were first processed using a rolling-ball background subtraction algorithm (FIJI)³⁸ with a 200 pixel radius. Subsequently, maximum intensity Z-projections of these images were generated. Spots were then localized and counted using a code developed by the Raj lab and available online (http://rajlab.seas.upenn.edu/StarSearch/launch.html). This image analysis was performed for FIGS. 1C-E, FIGS. 2A-C, FIG. FIGS. 5A-E, FIGS. 6A-G, FIGS. 7A-E, FIGS. 9A-B, 11A-C.

Analysis of Expansion Isotropy

smFISH images before and after expansion of TOP2A was rigidly aligned via two control points using the FIJI plugin Turboreg³⁹. Spots were localized and counted via a custom spot counting Matlab code developed by the Raj lab (complete source code and instructions can be found at https://bitbucket.org/arjunrajlaboratory/rajlabimagetools/wiki/Home). Length measurements were performed among all pairs of points before expansion and the corresponding pairs of points after expansion via a custom Matlab script. Measurement error was defined as the absolute difference between the before and after expansion length measurements (FIG. 8C).

Re-embedding of Expanded Gels in Acrylamide Matrix

For serial staining in cells, expanded gels were re-embeded in acrylamide to stabilize the gels in the expanded state. Briefly: gels were expanded in water and cut manually to ˜1 mm thickness with a stainless steel blade. Cut gels were incubated in 3% acrylamide, 0.15% N,N′-Methylenebisacrylamide with 0.05% APS, 0.05% TEMED and 5 mM Tris ph 10.5 for 20 minutes on a shaker. There is a ˜30% reduction in gel size during this step. Excess solution is removed from the gels and the gels are dried with light wicking from a laboratory wipe. Gels are placed on top of a bind-silane treated (see below) coverslip or glass bottom plate with a coverslip placed on top of the gels before moving into a container and purged with nitrogen. The container is moved to a 37° C. incubator for gelation for 1.5 hours.

Staining of Re-Embedded Gels

Re-embeded staining of gels were performed with exact conditions as described above for expanded gels, except post-hybridization washes were changed to twice with wash buffer (10% formamide), 60 minutes per wash.

Probes were removed for multiple rounds of hybridization via treatment with DNAse I or 100% formamide. For DNAse I, samples were treated with DNAse I at 0.5 U/μL for 6 hours at RT. For formamide stripping, samples were treated with 100% formamide at 6 hours at 37C.

Bind-silane Treatment of Coverslips

Coverslips and glass bottom 24 well plates were treated with Bind-Silane, a silanization reagent which incorporates acryloyl groups onto the surface of glass to perform in free radical polymerization. Briefly, 5 μL of Bind-Silane reagent was diluted into 8 mL of ethanol, 1.8 mL of ddH₂O and 200 μL of acetic acid. Coverslips and glass bottom 24 well plates were washed with ddH₂O followed by 100% ethanol, followed by the diluted Bind-Silane reagent. After a brief wash with the diluted Bind-Silane reagent, the cover-slip was dried, then washed with 100% ethanol, and then dried again. Coverslips were prepared immediately before use.

Probe Design for HCR-FISH

Probe sequences and accession numbers for mRNA targets can be found in Table 4. Probes were designed for HCR-FISH by tiling the CDS of mRNA targets with 22-mer oligos spaced by 3-7 bases. HCR initiators were appended to tiled sequences via a 2 base spacer (AA). For 2 color probe-sets, even and odd tiled probes were assigned different HCR-initiators to allow for amplification in different color channel.

RNA FISH with Hybridization Chain Reaction (HCR) Amplification

Gelled samples were incubated with wash buffer (20% formamide, 2× SSC) for 30 mins at room temperature and hybridized with HCR initiator tagged FISH probes in hybridization buffer (20% formamide, 10% dextran sulfate, 2× SSC) overnight at 37° C. Following hybridization, samples were washed twice with wash buffer, 30 mins per wash, and incubated with 1× PBS for 2 hrs at 37° C. Subsequently, samples were incubated with 1× PBS for at least 6hrs at room temperature. Before HCR amplification, hybridized samples were pre-incubated with amplification buffer (10% dextran sulfate, 5× SSC, 0.1% Tween 20) for 30 mins. To initiate amplification, HCR hairpin stocks (Alexa 456 and Alexa 647 fluorophores) at 3 μM were snap-cooled by heating to 95° C. for 90 seconds, and leaving to cool at room temperature for 30 mins. Gelled samples were then incubated with HCR hairpins diluted to 60 nM in amplification buffer for 3 hrs at room temperature. After amplification, gels were washed with 5× SSCT (5× SSC, 0.1% Tween 20) twice with one hour per wash.

Imaging of Cultured Cells using ExFISH

Both cultured cells as well as LabelX treated and expanded cultured cells were imaged on a Nikon Ti-E epifluorescence microscope with a SPECTRA X light engine (Lumencor), and a 5.5 Zyla sCMOS camera (Andor), controlled by NIS-Elements AR software. For FIGS. 1C, 1D, and FIGS. 6A-G, FIGS. 7A-E, and FIGS. 8-D a 40×1.15 NA water immersion objective was used. For all other experiments with cultured cells, a 60×1.4 NA oil immersion objective was used.

For imaging smFISH probes labeled with fluorophores, the following filter cubes (Semrock, Rochester, N.Y.) were used: Alexa 488, GFP-1828A-NTE-ZERO; Quasar 570, LF561-B-000; Alexa 594, FITC/TXRED-2X-B-NTE; Atto 647N, Cy5-4040C-000.

Imaging of Expanded Brain Slices

For epifluorescence imaging of brain sections before and after expansion (FIGS. 3A-E) and to quantify expansion factors of tissue slices specimens were imaged on a Nikon Ti-E epifluorescence microscope with a 4×0.2 NA air objective, a SPECTRA X light engine (Lumencor), and a 5.5 Zyla sCMOS camera (Andor), controlled by NIS-Elements AR software.

Post-expansion confocal imaging of expanded brain tissue was performed on an Andor spinning disk (CSU-X1 Yokogawa) confocal system with a 40×1.15 NA water objective (FIGS. 3F-K, FIGS. 13A-G) on a Nikon TI-E microscope body. GFP was excited with a 488 nm laser, with 525/40 emission filter. Alexa 546 HCR amplicons were excited with a 561 nm laser with 607/36 emission filter. Alexa 647 amplicons were excited with a 640 nm laser with 685/40 emission filter.

Gels were expanded in with 3 washes, 15 minutes each of 0.05× SSC. The expansion factor can be controlled with the salt concentration. It was found that 0.05× SSC gives 3× expansion, while still giving enough salt for hybridization stability. To stabilize the gels against drift during imaging following expansion, gels were placed in glass bottom 6 well plates with all excess liquid removed. If needed, liquid low melt agarose (2% w/w) was pipetted around the gel and allowed to solidify, to encase the gels before imaging.

Lightsheet imaging was performed on a Zeiss Z.1 lightsheet microscope. Briefly, the sample was fixed on a custom-made plastic holder using super glue and mounted on the freely rotating stage of the Z.1 lightsheet. Lightsheets were generated by two illumination objectives (5×, NA 0.1), and the fluorescence signal detected by a 20× water immersion objective (NA 1.0). Both lightsheets were used for data collection. The image volume dimensions of a single tile were 1400×1400×1057 pixels, with a voxel size of 227 nm laterally and 469 nm axially. The laserlines used for excitation were 488 nm, 561 nm and 638 nm. The individual laser transmissions were set to 5%, with the maximum output of 50 mW (488 nm and 561 nm) and 75 mW (638 nm). Optical filters used to separate and clean the fluorescence response included a Chroma T5601 pxr as a dichroic, and a Chroma 59001 m for GFP and 59007 m for Alexa 546 and Alexa 647. Two PCO.Edge 5.5 m sCMOS cameras were used to capture two fluorescence channels simultaneously. Tiled datasets were taken with the Zeiss ZEN Software, and subsequently merged and processed with FIJI, Arivis Vision4D and Bitplane Imaris.

Two Color Analysis in Slices

A sliding window averaging (or minimization) scheme in Z (3 optical sections) was used to suppress movement artifacts before spot detection processing. RNA puncta were detected via a custom 3D spot counting Matlab code developed by the Raj lab; complete source code and instructions can be found at https://bitbucket. org/arjunrajlaboratory/rajlabimagetools/wiki/Home.

Spot centroids were extracted from both color channels, and spots were determined to be co-localized if their centroids were within a 3 pixel radius in the x,y dimensions and a 2 pixel radius in the z dimension.

HCR Reversal via Toe-Hold Mediated Strand Displacement

HCR amplification commences upon the addition of two HCR metastable amplifier hairpins. We designed a pair of HCR amplifiers, B2H1T and B2H2 (see below for sequence), where B2H1T bears a 6 bp toe-hold for strand displacement. To initiate HCR amplification, aliquots of these amplifiers at 3 μM were snap-cooled by heating to 95° C. for 90 seconds, and leaving to cool at room temperature for 30 mins. Gelled samples were then incubated with HCR hairpins diluted to 60 nM in amplification buffer for 3hrs at room temperature. After amplification, gels were washed with 5× SSCT (5× SSC, 0.1% Tween 20) twice with one hour per wash. Subsequently, HCR reversal was initiated by the addition of a displacement strand (see below for sequence) at 200 nM in 5× SSCT.

B2H1T: ggCggTTTACTggATgATTgATgAggATTTACgAggAgCTCAgTCCATCC TCgTAAATCCTCATCAATCATCAAATAG. B2H2: /5′-Alexa546-C12/ CCTCgTAAATCCTCATCAATCATCCAgTAAACCgCCgATgATTgATgAgg ATTTACgAggATggACTgAgCT. Displacement Strand: CTATTTGATGATTGATGAGGATTTAcGAGGATGGAcTGAGcT.

REFERENCES:

-   1. Chen, F., Tillberg, P. W. & Boyden, E. S. Expansion microscopy.     Science (80-.). 347, 543-548 (2015). -   2. Femino, A. M., Fay, F., Fogarty, K. & Singer, R. Visualization of     Single RNA Transcripts in Situ. Science (80,). 280, 585-590 (1998). -   3. Levsky, J. M. & Singer, R. H. Fluorescence in situ hybridization:     past, present and future. J. Cell Sci. 116, 2833-2838 (2003). -   4. Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A.     & Tyagi, S. Imaging individual mRNA molecules using multiple singly     labeled probes. Nat. Methods 5, 877-9 (2008). -   5. Choi, H. M. T. et al. Programmable in situ amplification for     multiplexed imaging of mRNA expression. Nat. Biotechnol. 28, 1208-12     (2010). -   6. Choi, H. M. T., Beck, V. A. & Pierce, N. A. Next-Generation in     Situ Hybridization Chain Reaction: Higher Gain, Lower Cost, Greater     Durability. ACS Nano 8, 4284-4294 (2014). -   7 Cajigas, I. J. et al. The local transcriptome in the synaptic     neuropil revealed by deep sequencing and high-resolution imaging.     Neuron 74, 453-66 (2012). -   8. Wang, F. et al. RNAscope: A novel in situ RNA analysis platform     for formalin-fixed, paraffin-embedded tissues. J. Mol. Diagnostics     14, 22-29 (2012). -   9. Tillberg, P. W. et al. Expansion Microscopy of Biological     Specimens with Protein Retention. Nat. Biotechnol. -   10. Chozinski, T. J. et al. Expansion microscopy with conventional     antibodies and fluorescent proteins. Nat. Methods (2016).     doi:10.1038/nmeth.3833 -   11. Engreitz, J. M. et al. The Xist 1ncRNA exploits     three-dimensional genome architecture to spread across the X     chromosome. Science 341, 1237973 (2013). -   12. Panning, B., Dausman, J. & Jaenisch, R. X chromosome     inactivation is mediated by Xist RNA stabilization. Cell 90, 907-16     (1997). -   13. Plath, K., Mlynarczyk-Evans, S., Nusinow, D. A. & Panning, B.     Xist RNA and the mechanism of X chromosome inactivation. Annu. Rev.     Genet. 36, 233-78 (2002). -   14. Mito, M., Kawaguchi, T., Hirose, T. & Nakagawa, S. Simultaneous     multicolor detection of RNA and proteins using super-resolution     microscopy. Methods (2015). doi:10.1016/j.ymeth.2015.11.007 -   15. Clemson, C. M. et al. An architectural role for a nuclear     noncoding RNA: NEAT1 RNA is essential for the structure of     paraspeckles. Mol. Cell 33, 717-26 (2009). -   16. Lieberman-Aiden, E. et al. Comprehensive mapping of long-range     interactions reveals folding principles of the human genome. Science     326, 289-93 (2009). -   17. Lubeck, E. & Cai, L. Single-cell systems biology by     super-resolution imaging and combinatorial labeling. Nat. Methods 9,     743-8 (2012). -   18. Lubeck, E., Coskun, A. F., Zhiyentayev, T., Ahmad, M. & Cai, L.     Single-cell in situ RNA profiling by sequential hybridization. Nat.     Methods 11, 360-1 (2014). -   19. Chen, K. H., Boettiger, A. N., Moffitt, J. R., Wang, S. &     Zhuang, X. Spatially resolved, highly multiplexed RNA profiling in     single cells. Science (80-.). 348, aaa6090-aaa6090 (2015). -   20. Beliveau, B. J. et al. Versatile design and synthesis platform     for visualizing genomes with Oligopaint FISH probes. Proc. Natl.     Acad. Sci. U. S. A. 109, 21301-6 (2012). -   21. Feng, G. et al. Imaging neuronal subsets in transgenic mice     expressing multiple spectral variants of GFP. Neuron 28, 41-51     (2000). -   22. Lein, E. S. et al. Genome-wide atlas of gene expression in the     adult mouse brain. Nature 445, 168-76 (2007). -   23. Huisken, J., Swoger, J., Bene, F. Del, Wittbrodt, J. &     Stelzer, E. H. K. Optical Sectioning Deep Inside Live Embryos by     Selective Plane Illumination Microscopy. Science 305, 1007-1009     (2004). -   24. Batish, M., van den Bogaard, P., Kramer, F. R. & Tyagi, S.     Neuronal mRNAs travel singly into dendrites. Proc. Natl. Acad. Sci.     109, 4645-4650 (2012). -   25. Cabili, M. N. et al. Localization and abundance analysis of     human lncRNAs at single-cell and single-molecule resolution. Genome     Biol. 16, 20 (2015). -   26. Zhang, D. Y. & Seelig, G. Dynamic DNA nanotechnology using     strand-displacement reactions. Nat. Chem. 3, 103-113 (2011). -   27. Lee, J. H. et al. Highly Multiplexed Subcellular RNA Sequencing     in Situ. Science (80-.). 343, 1360-1363 (2014). -   28. Ke, R. et al. In situ sequencing for RNA analysis in preserved     tissue and cells. Nat. Methods 10, 857-60 (2013). -   29. Shah, S. et al. Single-molecule RNA detection at depth via     hybridization chain reaction and tissue hydrogel embedding and     clearing. Development In Review, (2016). -   30. Bruchez, M. et al. Semiconductor nanocrystals as fluorescent     biological labels. Science 281, 2013-6 (1998). -   31. Fouz, M. F. et al. Bright Fluorescent Nanotags from Bottlebrush     Polymers with DNA-Tipped Bristles. ACS Cent. Sci. 1, 431-438 (2015). -   32. Steward, O., Wallace, C. S., Lyford, G. L. & Worley, P. F.     Synaptic activation causes the mRNA for the leg Arc to localize     selectively near activated postsynaptic sites on dendrites. Neuron     21, 741-751 (1998). -   33. Buckley, P. T. et al. Cytoplasmic Intron Sequence-Retaining     Transcripts Can Be Dendritically Targeted via ID Element     Retrotransposons. Neuron 69, 877-884 (2011). -   34. Steward, 0. & Schuman, E. M. Compartmentalized synthesis and     degradation of proteins in neurons. Neuron 40, 347-359 (2003). -   35. Buxbaum, A. R., Wu, B. & Singer, R. H. Single -Actin mRNA     Detection in Neurons Reveals a Mechanism for Regulating Its     Translatability. Science (80-.). 343, 419-422 (2014). -   36. Jung, H., Yoon, B. C. & Holt, C. E. Axonal mRNA localization and     local protein synthesis in nervous system assembly, maintenance and     repair. Nat. Rev. Neurosci. 13, 308-24 (2012). -   37. Raj, A. & Tyagi, S. Detection of individual endogenous RNA     transcripts in situ using multiple singly labeled probes. Methods in     enzymology 472, (Elsevier Inc., 2010). -   38. Schindelin, J. et al. Fiji: an open-source platform for     biological-image analysis. Nat. Methods 9, 676-82 (2012). -   39. Thévenaz, P., Ruttimann, U. E. & Unser, M. A pyramid approach to     subpixel registration based on intensity. IEEE Trans. Image Process.     7,27-41 (1998).

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. 

What is claimed is:
 1. A method for in situ genomic and transcriptomic assessment of target nucleic acids present in a biological sample comprising the steps of: a) treating the biological sample with a small molecule linker capable of linking to at least one target nucleic acid and to a swellable material; b) embedding the biological sample wherein the small molecule linker is bound to the at least one target nucleic acid in the biological sample and to the swellable material; c) subjecting the biological sample to a physical disruption method; d) swelling the swellable material to form an expanded biological sample; e) providing at least one oligonucleotide complementary to the at least one target nucleic acid, wherein the at least one oligonucleotide hybridizes to the at least one target nucleic acid; and f) genomically or transcriptomically assessing the expanded biological sample.
 2. The method according to claim 1, wherein the small molecule linkers are attached to target nucleic acids via a chemical reactive group capable of covalently binding the target nucleic acid.
 3. The method according to claim 1, wherein the small molecule linker is labeled.
 4. The method according to claim 1, wherein the at least one oligonucleotide is labeled.
 5. The method according to claim 1, wherein embedding the biological sample in a swellable material comprises permeating the biological sample with a composition comprising precursors of a swellable polymer and forming a swellable polymer in situ.
 6. The method according to claim 1, wherein the at least one target nucleic acid is anchored to the swellable material.
 7. The method according to claim 1, wherein the physical disruption method is an enzymatic digestion.
 8. The method according to claim 1, wherein the target nucleic acids are DNA and/or RNA.
 9. The method according to claim 3, wherein the expanded biological sample expresses one or more labeled target nucleic acids.
 10. The method according to claim 1, further comprising the additional step of buffering the expanded sample.
 11. The method according to claim 10, further comprising the additional step of re-embedding the buffered expanded biological sample in a non-swellable material.
 12. The method according to claim 11, further comprising the step of removing the at least one oligonucleotide complementary to the at least one target nucleic acid.
 13. The method according to claim 12, wherein the steps of providing at least one oligonucleotide, genomically or transcriptomically assessing the expanded biological sample and removing the at least one oligonucleotide are repeated so as to allow serial or sequential genomic or transcriptomic assessments of the expanded biological sample.
 14. The method of claim 12, wherein removing the at least one oligonucleotide which is hybridized to the at least one target nucleic acid comprises formamide and high temperatures.
 15. The method of claim 12, wherein removing the at least one oligonucleotide which is hybridized to the at least one target nucleic acid comprises endonucleases that specifically digest the at least one oligonucleotide. 